Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intestinal cells from chicken embryos were grown in chemically defined, serum-free medium. The majority of cultured cells exhibits an epithelial-like morphology. As demonstrated by indirect immunofluorescence, the epithelial cells, and not the contaminating fibroblasts, express Calbindin-D28K only after 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, is added to the culture medium. The highly sensitive reverse transcriptase-polymerase chain reaction shows that both Calbindin-D28K mRNA and the corresponding primary unprocessed transcripts (pre-mRNA) are dramatically increased in cultured intestinal cells treated with 1,25-dihydroxyvitamin D3, thus indicating that Calbindin-D28K is induced by the increased rate of transcription of the corresponding gene.
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PMID:Induction of Calbindin-D28K by 1,25-dihydroxyvitamin D3 in cultured chicken intestinal cells. 157 13

The calbindin D9k (CaBP9k) gene is under strict estrogen control in the rat uterus. This tissue contains two CaBP9k messenger RNA (mRNA) species. We have used primer extension analysis, reverse transcriptase associated with polymerase chain reaction, and RNase H digestion to show that these two mRNA species have the same structural features, including 5'- and 3'-ends, and poly(A) tail length. Our results suggest that the difference in electrophoretic mobilities of the two mRNA species might be due to interaction with another factor. We also analyzed the imperfect estrogen-responsive element (ERE) present on the first 5'-splice site of the rat CaBP9k gene. The oligonucleotide corresponding to the CaBP9k ERE was cloned in the plasmid pBLCAT2 (where the thymidine kinase promoter governs the expression of the chloramphenicol acetyl transferase gene) and transfected into MCF7 cells. This CaBP9k ERE was found to be a hormone-inducible enhancer that worked in an orientation-independent manner on a heterologous promoter and was functional at physiological hormone concentrations. One CaBP9k ERE conferred only weak (about 2-fold) estrogen induction, but two EREs cloned in tandem were strongly synergistic (14- to 16-fold). The CaBP9k ERE also bound to the partially purified estrogen receptor (ER) and to ER expressed in COS cells by gel shift assay. Methylation interference showed that all the guanine residues in both half-sites of the CaBP9k ERE were protected by ER binding. Thus, ER binds to the CaBP9k ERE in a way similar to other EREs. The gel shift assay results indicate that the strong synergistic effect of two EREs cloned in tandem is not due to cooperative binding between the two elements. As the CaBP9k gene is under strong estrogenic control in the uterus in vivo, the imperfect CaBP9k ERE may cooperate with another trans-acting factor to become fully efficient.
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PMID:Calbindin-D9k gene expression in the uterus: study of the two messenger ribonucleic acid species and analysis of an imperfect estrogen-responsive element. 750 2

We have identified and quantified specific mRNAs in the human colonic carcinoma cell line Caco-2 by reverse transcriptase-polymerase chain reaction. Initial examination revealed that like rat duodenal mucosa, Caco-2 cells possessed mRNA for the vitamin D receptor. Using primers for human calbindin we found a 237-bp PCR product in Caco-2 cell RNA, but not from rat duodenal RNA. Primers for rat calbindin did not amplify calbindin mRNA in Caco-2 RNA, confirming a high degree of mismatch between rat and human sequences. 1,25(OH)2 vitamin D3 treatment (10 nM) significantly elevated calbindin mRNA levels 50% by 12 h, with maximal levels occurring by 48 h (fivefold elevation). Increasing concentrations of 1,25(OH)2 vitamin D3 (from 15 pM to 100 nM) caused progressive increases in calbindin mRNA levels following 48 h of treatment. Elevated calbindin mRNA levels were associated with enhancement of transcellular calcium transport. Our results are the first demonstration of vitamin D-regulated calbindin mRNA in a human intestinal cell line.
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PMID:Identification of calbindin D-9k mRNA and its regulation by 1,25-dihydroxyvitamin D3 in Caco-2 cells. 831 49

Calbindin D28k has been reported to be involved in the transcellular calcium transport along the rat distal tubule. It has also been shown that chronic metabolic acidosis (CMA) induces significant hypercalciuria. The present study investigated whether CMA affects the mRNA and the protein expression of calbindin D28k along isolated distal tubule (DT) of rats. The animals were made acidotic by adding 0.28 mol/L NH4Cl to the drinking water for 7 d. This maneuver was associated with an increase in plasma ionized calcium. Inulin clearance experiments demonstrated that metabolic acidosis did not affect GFR, but it significantly increased both total and fractional urinary calcium excretion. To define the role of calbindin D28k, total RNA was extracted from DT, identified, and microdissected from collagenase-treated kidneys. cDNA was synthesized from RNA using reverse transcriptase and oligo(dT)(12-18) primers. Calbindin D28k mRNA abundance was semiquantified by a competitive reverse transcription-PCR, using an internal standard of cDNA that differed from the wild-type calbindin D28k by a deletion of 86 bp. The reverse transcription-PCR was performed starting from the same amount of total RNA. For each set of experiments, control and acidotic rats were studied in parallel. The identity of the DT was further verified by the presence of the thiazide-sensitive NaCl cotransporter (rTSC1) mRNA. Calbindin D28k mRNA abundance was 0.89 +/- 0.21 amol/ng total RNA in DT of CMA rats (n = 5) compared with 0.30 +/- 0.12 amol/ng total RNA of control rats (n = 5) (P < 0.05). Using specific rabbit polyclonal anti-calbindin D28k antibody, Western blotting was performed starting from thin slices of outer cortex. Densitometric analysis revealed that in acidotic rats (n = 7) there was a 17 +/- 5% (P < 0.05) increase in calbindin D28k protein abundance compared with controls (n = 7). These results indicate that in the rat, ammonium chloride loading induces an increase in filtered ionized calcium load that is associated with a significant upregulation of calbindin D28k both at the mRNA and protein level. These last effects will help to reduce the concomitant hypercalciuria, thus mitigating the consequence of CMA on calcium metabolism.
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PMID:Effect of chronic metabolic acidosis on calbindin expression along the rat distal tubule. 1066 27

A significant contribution of the forestomachs in net calcium (Ca2+) absorption from the gastrointestinal tract has been postulated from in vivo and in vitro studies in different ruminant species. However, the potential role of vitamin D3 and its metabolites in controlling these mechanisms is still under discussion. It was therefore the aim of the present study to investigate the effectiveness of treatment with vitamin D3 in stimulating active Ca2+ absorption from sheep rumen. Four mature, non-lactating, non-pregnant sheep that had been treated 7 and 4 days before the Ca2+ flux rate measurements with intramuscular injections of 300000 IU of vitamin D3 each in aqueous solution were used. Two female and three male placebo-treated sheep served as controls. To characterize the effects of vitamin D3 application on plasma parameters the time courses of total calcium, inorganic phosphate, calcitriol and intact parathyroid hormone (iPTH) were recorded. In vitro studies of unidirectional Ca2+ flux rates across isolated, intact rumen wall epithelia were carried out by applying the Ussing-chamber technique. Western blot analysis and reverse transcriptase-polymerase chain reaction analysis (RT-PCR) were applied to identify vitamin D receptors (VDR) in ruminal and jejunal tissues. In addition, Western blot analysis for qualitative examination of epithelial calbindin D9k levels was carried out in these tissues. Total calcium and phosphate levels in plasma were not significantly affected treatment with vitamin whereas calcitriol concentrations significantly increased by about 130 and 63% after the first and second application, respectively. In contrast, iPTH tended to decrease by about 60% indicating regulatory effects of calcitriol on systemic Ca homeostasis. The Ca2+ flux rate measurements in Ussing-chambers revealed significant net Ca2+ absorption indicating the contribution of active mechanisms for Ca2+ transport in rumen epithelia. This, however, was not significantly affected by increased calcitriol concentrations in plasma. Western blot analysis on the basis of a human recombinant VDR protein and RT-PCR clearly indicated the presence of VDR in ruminal and jejunal epithelia, but, in contrast to jejunum, this was not reflected by respective amounts of calbindin-D9k in ruminal tissues. The results suggest the absence of classical calbindin-D9k-mediated mechanisms for active Ca2+ transport in sheep rumen.
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PMID:No effect of vitamin D3 treatment on active calcium absorption across ruminal epithelium of sheep. 1155 93

The effects of GABA in the CNS are mediated by three different GABA receptors: GABA(A), GABA(B) and GABA(C) receptors. GABA(A) and GABA(B) receptors, but not yet GABA(C) receptors, have been demonstrated in the enteric nervous system, where GABA has been proposed to be a transmitter. The purpose of this study was to determine whether GABA(C) receptors are present and thus may play a role in mediating the effects of GABA in the myenteric plexus of the rat gastrointestinal tract. We examined the expression of the three known GABA(C) receptor subunits, rho1, rho2 and rho3, in the rat duodenum, ileum and colon using the reverse transcriptase-polymerase chain reaction. We determined the localization of GABA(C) receptors in the myenteric plexus of these regions using two different antisera directed against GABA(C) receptor subunits. The polymerase chain reaction revealed that all three subunits were expressed in the gastrointestinal tract. When the layers of the intestine were separated and the layer containing myenteric neurons was assayed, the rho3 subunit was found in the ileum and colon, whereas rho1 was expressed in the duodenum and weakly in the colon and rho2 was expressed in the ileum. Immunocytochemistry revealed numerous labeled neurons in the myenteric plexus of each region. Colocalization showed that a large proportion of calbindin plus calretinin immunoreactive neurons (intrinsic primary afferent neurons) were immunoreactive for the GABA(C) receptor, and that 56% of nitric oxide synthase immunoreactive neurons (inhibitory motor neurons) exhibited the receptor. These results indicate that GABA(C) receptors of differing subunit compositions are expressed by neurons in the rat gastrointestinal tract. The effects of GABA on intrinsic sensory and on inhibitory motor neurons are likely to be mediated in part through GABA(C) receptors.
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PMID:Gene expression and localization of GABA(C) receptors in neurons of the rat gastrointestinal tract. 1174 57

The epithelial Ca2+ channel, ECaC1, is primarily expressed in the apical membrane of vitamin D-responsive tissues. This study characterizes for the first time the presence of this novel channel in pancreatic tissue by reverse transcriptase-polymerase chain reaction and immunohistochemistry. In addition, the expression of ECaC1 was investigated in an animal model for Type 2 diabetes mellitus, the Zucker diabetic fatty (ZDF) rat. Identical staining patterns for ECaC1 and insulin were observed, whereas no co-localization of ECaC1 with glucagon was found. ECaC1, insulin, and prohormone convertase 1 (a neuroendocrine endoprotease expressed in secretory granules) showed a similar punctate staining. ECaC1 co-localized with the Ca2+ binding protein calbindin-D(28K) in the beta-cells. Furthermore, in contrast to wild-type rats, in ZDF rats aging led to a progressive decrease in both insulin and ECaC1 staining. Plasma 1,25-dihydroxyvitamin D3 levels were similar in both control and ZDF rats and decreased with aging. Taken together, our findings indicate that this novel Ca2+ channel may play a role in the regulation of endocrine Ca2+ homeostasis.
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PMID:Expression of the novel epithelial Ca2+ channel ECaC1 in rat pancreatic islets. 1201 95

Calbindin-D28k (CaBP28k) belongs to a large class of eucaryotic proteins that bind calcium (Ca2+) to a specific helix-loop-helix structure. To date, this protein was mainly linked to brain, kidneys, and pancreas. Here, we demonstrate for the first time the existence of CaBP8k in the human placental trophoblasts of the human term placenta. Placental Ca2+ transfer from maternal to fetus is crucial for fetal development, although the biochemical mechanisms responsible for this process are largely unknown. In the current study, we have investigated the 45Ca2+ uptake by human trophoblast cells in correlation with the expression CaBP28k. The expression of CaBP28k was determined by Northern blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), immunochemistry, and Western blot analysis. Indeed, Northern blot analysis revealed the presence of a CaBP28k transcript in syncytiotrophoblasts, cytotrophoblast cells, and HEK-293 cells. This was further confirmed by RT-PCR analysis followed by sequencing. In addition, anti-CaBP28k labeling was associated with cytotrophoblast and syncytiotrophoblast tissues in placental tissue sections and in vitro cultured cells. The presence of CaBP28k protein in these cells was confirmed by Western blotting. Cytotrophoblast cells isolated from human term placenta showed differentiation into syncytiotrophoblasts in culture according to the increase in hCG secretion. Both Ca2+ uptake and hCG secretion by trophoblasts increased gradually and were high at Day 4. Taken together, these data suggest that CaBP28k may play a role in Ca2+ transport or cell development in human trophoblast possibly trough Ca2+ buffering.
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PMID:Expression of calbindin-D28k (CaBP28k) in trophoblasts from human term placenta. 1260 74

Motilin, a 22-amino acid gastrointestinal peptide, and ghrelin, the natural ligand of the growth hormone secretagogue receptor, form a new group of structurally related peptides. Several lines of evidence suggest that motilin and ghrelin are involved in the control of gastrointestinal motility by the activation of receptors on enteric neurons. The aim of this study was to look for the existence of motilin, ghrelin, and their respective receptors in the myenteric plexus of the guinea pig. We used longitudinal muscle/myenteric plexus (LMMP) preparations and cultures of myenteric neurons of the guinea pig ileum, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR). Most of the motilin-immunoreactive (IR; 72.8%) and motilin receptor-IR (68.9%) neurons were also positive for neuronal nitric oxide synthase (nNOS), 72.8% and 68.9%, few for choline acetyl transferase (ChAT), 11.4% and 11.9%, respectively. In contrast, ghrelin was mainly colocalized with ChAT (72.2%), and only 3.6% of ghrelin-positive cells showed nNOS-IR in the LMMP. Neither motilin nor the motilin receptor or ghrelin colocalized with calbindin. RT-PCR studies revealed motilin, ghrelin, and ghrelin receptor mRNA transcripts in LMMP preparations and in cultured myenteric neurons. In conclusion, this study, for the first time, provides direct evidence for the existence of motilin and ghrelin in myenteric neurons and suggests that both peptides may play a role in the activation of the enteric nervous system and hence in the regulation of gastrointestinal motility.
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PMID:Evidence for the presence of motilin, ghrelin, and the motilin and ghrelin receptor in neurons of the myenteric plexus. 1554 49

Conjugated linoleic acid (CLA) has been shown to enhance paracellular and transcellular Ca transport across human intestinal-like Caco-2 cell monolayers. The mechanisms of action, however, are still unclear. Therefore, this study investigated the molecular mechanisms underlying CLA-induced stimulation of Ca transport by use of preliminary microarray data together with more detailed and comprehensive quantitative reverse transcriptase-PCR analysis. While molecular expression of junctional adhesion molecule (JAM), ZO-2, ZO-3, claudin 2 and claudin 3 were unaltered, ZO-1, occludin, and claudin 4 were all up-regulated (1.6, 1.6, 2.4-fold, respectively; P<0.001-0.01) and claudin 1 down-regulated (2.5-fold; P<0.05) by trans-10, cis-12 CLA, which may underpin its effects on tight-junction function and paracellular Ca transport. On the other hand, expression of key genes involved in transcellular Ca transport (CaT1, ECaC1, calbindin D(9k), vitamin D receptor and PMCA) were unaffected by trans-10, cis-12 CLA. The mechanism by which CLA enhances transcellular Ca transport remains unclear.
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PMID:Conjugated linoleic acid enhances transepithelial calcium transport in human intestinal-like Caco-2 cells: an insight into molecular changes. 1665 Jul 47


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